1. Field of the Invention
The present invention relates to a method for identifying animals that are resistant or susceptible to diseases associated with intracellular parasites. More particularly, the present invention relates to the identification of a gene, called NRAMP1, associated with the susceptibility or resistance of an animal, such as an artiodactyla, to diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. Still more particularly, the present invention relates to the identification of specific sequences of the 3' untranslated region (3' UTR) of bovine NRAMP1 which associate with resistance or susceptibility to bovine brucellosis, tuberculosis, paratuberculosis and salmonellosis, and to the use of the general sequence patterns to identify artiodactyl animals containing those sequences in situ, allowing therefore the identification of animals predicted to be either resistant or susceptible to diseases associated with intracellular parasites.
2. General Background
Intracellular zoonotic bacterial diseases like brucellosis and tuberculosis cause significant losses in livestock industries despite widespread application of antimicrobials, vaccination, isolation and quarantine, test and slaughter, or a combination of these. The lack of success in eradicating infectious diseases of animals using these approaches indicates a need for a different strategy, such as the development of a means to identify genetic sequences associated with resistance and/or susceptibility, where such means could allow the identification of animals that are resistant or susceptible to disease. This could then allow the treatment, prophylactic or therapeutic, or elimination of susceptible animals, and the use of and/or selective breeding of resistant animals (see, for example, Templeton et al. 1988).
Diseases such as ruminant brucellosis, tuberculosis, paratuberculosis and salmonellosis cause an estimated $250,000,000 loss annually to the U.S.A. beef and dairy industry. Further, tuberculosis especially is a health threat to all ungulates including rare and endangered mammals. These are diseases for which the usual eradication programs have been long-term, expensive, and somewhat unsuccessful. For example, bovine tuberculosis was thought to be a disease of antiquity in 1970 but has re-emerged as an endemic disease in the El Paso, Tex. dairy herds. Outbreaks of bovine tuberculosis have been reported in the past 5 years in California, Idaho, Indiana, Louisiana, Missouri, Montana, Nebraska, New Mexico, New York, North Carolina, Pennsylvania, South Carolina, Texas, Wisconsin, and Virginia (Essey and Koller 1994; and Essey M. A. 1991).
Further, each of these specific diseases are zoonotic diseases which continually threaten the U.S. population. The benefit of cattle naturally resistant to these, and other diseases would be a key component of the preharvest pathogen reduction programs like the National Hazard Analysis Critical Control Point (HACCP) program proposed for farm use (Pierson, M. D. and Corlett, D. A., 1992; and Vanderzant, C., 1985). Further, it is desired that the approach used to control these diseases use natural resistance since it is environmentally compatible.
The only method currently available for the detection of artiodactyla resistant to brucellosis or tuberculosis is by a potent in vivo challenge with virulent Brucella abortus, Salmonella dublin, Mycobacterium paratuberculosis, or Mycobacterium bovis (Templeton and Adams 1996). Unfortunately, for this assay, the tested ungulates have to be euthanized in order to culture for the specific pathogen. Males challenged with B. abortus or M. bovis must be necropsied and cultured to determine if the bacterium has been cleared (resistant) or persists (susceptible). Nonpregnant females challenged with M. bovis must be necropsied and cultured to determine resistance or susceptibility. Although the gametes from both males and females can be stored frozen and used in a breeding-selection program to produce naturally resistant progeny with some success, this is both extremely expensive, and inefficient. The viability of frozen gametes and embryos is variable and a much lower birth rate occurs than with natural matings. Additionally, the breeding-selection program would be based on phenotypic selection (so-called mass selection) which is not as efficient as determining genotypes and selecting resistance associated with genetic sequences directly. (See, for example, Martin et al. 1994; and Dietrich et al. 1986).
The present invention solves these prior art problems by providing an efficient and reliable method for determining whether an animal, such as an artiodactyla, is susceptible or resistant to diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis.